Abstract: The autosomal-dominant hyper-IgE syndrome (HIES), caused by mutations in STAT3, is a rare primary immunodeficiency that predisposes to mucocutaneous candidiasis and staphylococcal skin and lung infections. This infection phenotype is suggestive of defects in neutrophils, but data on neutrophil functions in HIES are inconsistent. This study was undertaken to functionally characterize neutrophils in STAT3-deficient HIES patients and to analyze whether the patients` eosinophilia affects the neutrophil phenotype in S. aureus infection. Neutrophil functions and cell death kinetics were studied in eight STAT3-deficient patients. Moreover, the response of STAT3-deficient neutrophils to S. aureus and the impact of autologous eosinophils on pathogen-induced cell death were analyzed. No specific aberrations in neutrophil functions were detected within this cohort. However, the half-life of STAT3-deficient neutrophils ex vivo was reduced, which was partially attributable to the presence of eosinophils. Increased S. aureus-induced cell lysis, dependent on the staphylococcal virulence controlling accessory gene regulator (agr)-locus, was observed in STAT3-deficient neutrophils and upon addition of eosinophils. Accelerated neutrophil cell death kinetics may underlie the reported variability in neutrophil function testing in HIES. Increased S. aureus-induced lysis of STAT3-deficient neutrophils might affect pathogen control and contribute to tissue destruction during staphylococcal infections in HIES

Abstract: Fine particulate matter (PM2.5) can adversely affect human health. Emissions from residential energy sources have the largest impact on premature mortality globally, but their pathological and molecular implications on cellular physiology are still elusive. In the present study potential molecular consequences were investigated during long-term exposure of human bronchial epithelial BEAS-2B cells to PM2.5, collected from a biomass power plant. Initially, we observed that PM2.5 did not affect cellular survival or proliferation. However, it triggered an activation of the stress response p38 MAPK which, along with RhoA GTPase and HSP27, mediated morphological changes in BEAS-2B cells, including actin cytoskeletal rearrangements and paracellular gap formation. The p38 inhibitor SB203580 prevented phosphorylation of HSP27 and ameliorated morphological changes. During an intermediate phase of long-term exposure, PM2.5 triggered proliferative regression and activation of an adaptive stress response necessary to maintain energy homeostasis, including AMPK, repression of translational elongation, and autophagy. Finally, accumulation of intracellular PM2.5 promoted lysosomal destabilization and cell death, which was dependent on lysosomal hydrolases and p38 MAPK, but not on the inflammasome and pyroptosis. TEM images revealed formation of protrusions and cellular internalization of PM2.5, induction of autophagosomes, amphisomes, autophagosome-lysosomal fusion, multiple compartmental fusion, lysosomal burst, swollen mitochondria and finally necrosis. In consequence, persistent exposure to PM2.5 may impair epithelial barriers and reduce regenerative capacity. Hence, our results contribute to a better understanding of PM-associated lung and systemic diseases on the basis of molecular events

Abstract: The immune response to mycobacteria is characterized by granuloma formation, which features multinucleated giant cells as a unique macrophage type. We previously found that multinucleated giant cells result from Toll-like receptor-induced DNA damage and cell autonomous cell cycle modifications. However, the giant cell progenitor identity remained unclear. Here, we show that the giant cell-forming potential is a particular trait of monocyte progenitors. Common monocyte progenitors potently produce cytokines in response to mycobacteria and their immune-active molecules. In addition, common monocyte progenitors accumulate cholesterol and lipids, which are prerequisites for giant cell transformation. Inducible monocyte progenitors are so far undescribed circulating common monocyte progenitor descendants with high giant cell-forming potential. Monocyte progenitors are induced in mycobacterial infections and localize to granulomas. Accordingly, they exhibit important immunological functions in mycobacterial infections. Moreover, their signature trait of high cholesterol metabolism may be piggy-backed by mycobacteria to create a permissive niche

Abstract: Immunohistochemical staining of tissue sections is a vital technique in pathological diagnostics and theranostics. Several kinds of detection systems are available--each of them with their advantages and disadvantages. Here we present the results of a study assessing a prototype immunohistochemical detection technology (PIDT) for visualization of antigens in tissue sections. Different tumor tissues (n = 11) were stained with selected antibodies (n = 30) and a subset of these under different fixation conditions. The staining properties were assessed according to six staining quality parameters (signal distribution, intensity, tissue and slide background, acutance, clarity of details, and subcellular morphological details), and the results were compared with those of a well-established detection system (EnVision FLEX). Overall, both detection methods revealed good to optimal results regarding the evaluated parameters even under unfavorable fixation conditions. However, with the prototype detection technology a quicker turnaround time was reached primarily due to shorter primary antibody incubation times. Moreover, PIDT-stained tissues showed higher signal intensity and a uniform signal distribution over the tissue slide, still, with well-preserved tissue morphology and without impairing the gradation of staining intensity of different cell types. In particular, the prototype detection technology performed better in poorly or delayed fixed tissue. In situations where rapid and profound results are in demand, and particularly in the context of a small laboratory setting, this prototype detection technology could be a useful addition to the established detection systems

Abstract: Purpose About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. Methods Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. Results VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. Conclusions Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination

Abstract: Common variable immunodeficiency (CVID), characterized by recurrent infections, low serum class-switched immunoglobulin isotypes, and poor antigen-specific antibody responses, comprises a heterogeneous patient population in terms of clinical presentation and underlying etiology. The diagnosis is regularly associated with a severe decrease of germinal center (GC)-derived B-cell populations in peripheral blood. However, data from B-cell differentiation within GC is limited. We present a multiplex approach combining histology, flow cytometry, and B-cell receptor repertoire analysis of sorted GC B-cell populations allowing the modeling of distinct disturbances in GCs of three CVID patients. Our results reflect pathophysiological heterogeneity underlying the reduced circulating pool of post-GC memory B cells and plasmablasts in the three patients. In patient 1, quantitative and qualitative B-cell development in GCs is relatively normal. In patient 2, irregularly shaped GCs are associated with reduced somatic hypermutation (SHM), antigen selection, and class-switching, while in patient 3, high SHM, impaired antigen selection, and class-switching with large single clones imply increased re-cycling of cells within the irregularly shaped GCs. In the lymph nodes of patients 2 and 3, only limited numbers of memory B cells and plasma cells are formed. While reduced numbers of circulating post GC B cells are a general phenomenon in CVID, the integrated approach exemplified distinct defects during GC maturation ranging from near normal morphology and function to severe disturbances with different facets of impaired maturation of memory B cells and/or plasma cells. Integrated dissection of disturbed GC B-cell maturation by histology, flow cytometry, and BCR repertoire analysis contributes to unraveling defects in the essential steps during memory formation